Streptomyces Peucetius subsp. Caesius ATCC 27952 유래 aklavinone 11-hydroxylase 유전자의 대정균에서의 대량발현과 최적화

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Streptomyces Peucetius subsp. Caesius ATCC 27952 유래 aklavinone 11-hydroxylase 유전자의 대정균에서의 대량발현과 최적화
Woo Keon Min; Young Soo Hong; Yong Kyung Choe; Jung Joon Lee; Soon Kwang Hong
Bibliographic Citation
Korean Journal of (Applied) Microbiology & Biotechnology, vol. 26, no. 1, pp. 15-22
Publication Year
The dnrF gene, responsible for conversion of aklavinone to ε- rhodomycinone via C-11 hydroxylation, was mapped in the daunorubicin gene cluster of Streptomyces peucetius subsp. caesius ATCC 27952, close to drrAB, one of the anthracycline resistance genes. To characterize the enzymatic properties of the aklavinone 11-hydroxylase, the dnrF gene was overexpressed in Escherchia coli. The pET-22(+) plasmid which has the T7 promotor under the control of lacUV5 gene was used for the overexpression of the dnrF gene, and the recombinant plasmid pET213 that contains the dnrF gene linked to the T7 promotor of pET-22b(+) was introduced into the E. coli BL21. When the expression of the dnrF gene was induced by IPTG at the final concentration of 1 mM, the induced protein could be detected in SDS-PAGE only in insoluble precipitate. The insoluble protein was electroeluted from the gel and used for the preparation of antiserum in mice. Various culture conditions were tested to maximize the expression of the aklavinone 11-hydroxylase in soluble form. The enzymatic activity was checked by the bioconversion experiment, and the protein was confirmed by the SDS-PAGE and the Western blot analysis. From the analysis of the data, it was concluded that the culture induced with IPTG at the final concentration of 0.02 mM at 37°C yielded the best productivity of active form of enzyme.
overexpressionaklavinone 11-hydroxylasednrF Streptomyces peucetiusstreptomycesEscherichia coli
Korea Soc-Assoc-Inst
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Ochang Branch Institute > Chemical Biology Research Center > 1. Journal Articles
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